Upon its activation by growth factors (EGF, PDGF), PLC-gamma1 is phosphorylated on Tyr771, Tyr783 and Tyr 1253 by the corresponding receptor tyrosine kinase. Phospholipase activity induced by PDGF was unaffected by mutation of Tyr1253 to Phe, but mutation of Tyr771 decreased PDGF-induced inositol phosphates production (IPs) by about 50% and mutation of Tyr783 to Phe completely abolished the IPs response to PDGF. We prepared antibodies that specifically bind to each of the three phosphorylated site and used the phosphorylation-specific antibodies to study the kinetics of PLC-gamma1 phosphorylation in NIH 3T3 cells expressing both PDGFR and EGFR. Both EGF and PDGF caused phosphorylation in the following order: Tyr783 greater than Tyr1253 much greater than Tyr771. Kinetic and immunoblotting experiments suggest that a PLC-gamma1 molecule recruited to an activated growth factor receptor kinase is phosphorylated on the three sites without falling off the receptor in a processive reaction.The significance of the tyrosine phosphorylation of PLC-gamma1 was further investigated by analysing chromatographic behaviour of PLC-gamma1 (on Heparin column). The phosphorylated species of PLC-gamma1 are clearly separated from unphosphorylated PLC-gamma1. This separation is abolished by mutation of Tyr783 but not by mutation of Tyr771 or Tyr1253. This result indicates that the phosphorylated Tyr783 residue interacts intramolecularly with one of the two SH2 domains of PLC-gamma1, causing a major conformational change, or interacts intermolecularly with other SH2 domain-containing protein.Although EGF and PDGF induce similar extents of phosphorylation on the three sites, the amount of inositol phosphates produced in response to PDGF was much larger than that produced by EGF. Knowing that PtdIns 3,4,5-P3 is an activator of PLC-gamma, we compared the amounts of PtdIns 3,4,5-P3 produced in response to EGF and PDGF. The PtdIns 3,4,5-P3 production induced by PDGF was higher and last longer than the one evoked by EGF. Furthermore, PLC activity was diminished by pharmacological inhibition of PI-3K. These observations indicate that tyrosine phosphorylation of PLC-gamma1 is not sufficient for full activation of PLC-gamma1.